CN117357559A - Use of mitochondrial extracts for the treatment or/and prevention of diseases associated with kidney damage - Google Patents

Abstract

The invention discloses a use of mitochondrial extract for treating or/and preventing kidney injury related diseases, in particular to a method for effectively improving kidney injury related diseases and preventing the worsening of kidney injury related diseases by administering the mitochondrial extract provided by the invention to a subject suffering from kidney injury related diseases.

Description

Use of mitochondrial extract for treating and/or preventing diseases related to kidney damage

This application is the national application number given by the Patent Office of the State Intellectual Property Office as 202180014670.7, the international application number is PCT/CN2021/081686, the international application date is March 19, 2021, and the invention name is "mitochondria extract for the treatment of or / and prevention of diseases related to kidney damage" is a divisional application of the patent application.

Technical field

The present invention relates to the second use of mitochondrial extract, particularly the use of mitochondrial extract for treating or/and preventing diseases related to kidney damage.

Background technique

Mitochondria are organelles that exist in human cells and are used to provide ATP required for normal cell operation. Moreover, recent studies have pointed out that the increase and activation of mitochondria in cells will provide the energy needed for stem cell differentiation and contribute to the success of stem cells. differentiation. In other words, mitochondria play a very important role in human energy metabolism. For example, if mitochondria are defective, it will cause degenerative or aging-related diseases, such as brain degeneration, muscle weakness, muscle disease, etc.; and many studies currently point out that , if patients with Parkinson's disease or Alzheimer's disease caused by oxidative damage can maintain normal mitochondrial function or improve the body's antioxidant capacity, it will help worsen neurodegenerative diseases.

When the kidney tissue is damaged for more than 3 months, so that the structure or function of the kidney cannot be restored to its original function, it is called chronic kidney disease. Currently, clinical treatment is mainly based on drug treatment, supplemented by diet and lifestyle control. However, When chronic kidney disease gradually worsens over the course of the disease and the patient faces renal fibrosis and gradually loses kidney function, the patient needs to undergo hemodialysis or kidney transplantation to maintain life. This is not only a very uncomfortable process for the patient, but also has high medical costs. It is also a high burden. In other words, since there is currently no clear understanding of the pathogenesis and treatment methods of chronic kidney disease, it is impossible to provide effective clinical treatment for chronic kidney disease. Therefore, it is necessary to provide an effective treatment method for chronic kidney disease and kidney disease. Compositions or methods for fibrosis have become a clinical medical imperative.

Contents of the invention

The main purpose of the present invention is to provide a second use of mitochondrial extract, which can effectively improve or prevent kidney damage-related diseases, thereby achieving the effect of treating kidney disease or slowing down the progression of kidney disease.

Therefore, in order to achieve the above object, the present invention discloses the use of mitochondria for preparing a composition for preventing or/and treating diseases related to kidney damage. Specifically, by administering a certain amount of mitochondrial extract to a patient suffering from kidney disease. When treating individuals with damage-related diseases, it can improve the damage to kidney cells, thereby achieving the effect of treating or preventing the deterioration of kidney damage-related diseases.

In embodiments of the present invention, the kidney injury-related diseases are renal fibrosis, renal inflammation, chronic kidney disease, acute kidney disease, renal tubular injury, renal failure, prerenal injury, nephrogenic injury, and postrenal injury. , glomerulitis, pyelonephritis, nephrotic syndrome, uremia.

In one embodiment of the invention, the kidney damage-related disease has mitochondrial damage and at least one of the following symptoms: proteinuria, edema, oliguria, excessive urea nitrogen, excessive creatinine, abnormal uric acid, stones, and nephroglomeruli. Abnormal filtration rate.

In another embodiment of the invention, the mitochondria are isolated from a stem cell, such as adipose stem cells, CD34+ hematopoietic stem cells, mesenchymal stem cells, bone stem cells, umbilical cord stem cells, amniotic membrane stem cells, amniotic fluid stem cells, placental stem cells, iPS, neural stem cells.

In embodiments of the present invention, the dosage of mitochondria in the composition is 5-80 μg, and preferably the dosage of mitochondria in the composition is more than 40 μg.

The beneficial effects of the present invention are:

When mitochondrial damage occurs in kidney cells due to fibrosis, oxidative stress or an inflammatory environment, administration of the mitochondrial extract provided by the present invention or a composition containing it can effectively improve the mitochondrial damage of kidney cells, and thereby It can improve or treat kidney cell damage or related diseases.

Description of the drawings

Figure 1A shows the results of statistical analysis of the survival rate of kidney epithelial cells treated with different concentrations of hydrogen peroxide for 24 hours.

Figure 1B shows the results of statistical analysis of the survival rate of renal epithelial cells treated with different concentrations of hydrogen peroxide and then administered with different doses of mitochondrial precipitates.

Figure 2A shows the results of detecting and analyzing the amount of collagen secreted by renal epithelial cells after they were treated with different concentrations of glycation end products (AGEs-BSA) for different times.

Figure 2B shows the results of detecting and analyzing the amount of collagen secreted by renal epithelial cells after they were treated with different concentrations of AGEs-BSA and then administered with different doses of mitochondrial precipitates.

Figure 2C shows the results of detecting and analyzing the amount of collagen secreted by the renal epithelial cells after the renal epithelial cells were treated with different concentrations of hydrogen peroxide and then administered with different doses of mitochondrial precipitates.

Figure 3 shows the results of detecting and analyzing the mitochondrial damage of renal epithelial cells after they were treated with hydrogen oxide or AGEs-BSA respectively, and then administered with different doses of mitochondrial precipitate.

Detailed ways

The present invention provides a second use of mitochondrial extract, which means that by administering a certain amount of mitochondrial extract or a composition containing the same to an individual suffering from kidney damage-related diseases, the kidney damage-related diseases can be effectively improved, and To prevent the progression of kidney damage-related diseases.

Generally speaking, the dose of mitochondria provided by the present invention for administration to an individual is 5 to 80 μg, such as 5, 10, 15, 20, 25, 30, 40, 50, 60, 65, 70, 80 μg, etc., wherein, The dosage is preferably 15 to 40 μg; and, in order to achieve a better effect of treating or improving kidney disease, the mitochondria provided by the present invention can be combined with other components to prepare a composition, and the combined components are It is preferable to have growth factors, such as blood products containing growth factors, platelet-rich plasma (PRP), plasma, serum, platelet-rich fibrin (Platelet-Rich Fibrin), etc.

The "mitochondria extract" referred to in the present invention refers to mitochondria separated from a cell, and the separation technology or method used should be able to maintain the integrity of the mitochondrial structure and function. According to the present invention, those skilled in the art, separation Techniques or methods may be physical or chemical.

The "cells" referred to in the present invention refer to cells with mitochondria, such as adipose stem cells, mesenchymal stem cells, skeletal muscle cells, liver cells, kidney cells, fibroblasts, nerve cells, skin cells, blood cells, etc.

The "composition" referred to in the present invention can be a pharmaceutical composition, food, functional food, nutritional supplement, etc., and can be combined with different components according to the type, and has different dosage forms and different administration methods.

The "kidney damage-related diseases" referred to in the present invention are diseases caused by damage to kidney cells and have symptoms of mitochondrial damage, such as kidney fibrosis, kidney inflammation, kidney disease, acute kidney disease, renal tubular damage, and renal failure. , prerenal injury, nephrogenic injury, postrenal injury, glomerulitis, pyelonephritis, nephrotic syndrome, uremia, etc.

In the following, in order to confirm the efficacy of the mitochondrial extract provided by the present invention, several examples will be given and explained in detail with accompanying drawings.

The mitochondria used in the following examples are obtained from human adipose-derived stem cells, but there is no limitation that the mitochondria of the present invention can only be derived from human adipose-derived stem cells, which means that the mitochondria of the present invention can be obtained from any cell of the human body.

The mitochondrial dose used in the following examples is only an example. The mitochondrial dose of 15 μg is used as a low-dose representative, and 40 μg is used as a high-dose representative. They are not used to limit the technical features of the present invention, which means that the mitochondria provided by the present invention are used at a dose of 5 ~80 μg can achieve the desired effect of the present invention.

Example 1: Culture of kidney epithelial cell lines

Kidney epithelial cell lines were cultured in cell culture medium containing MEM-αEarl's salt and 5% fetal calf serum, and cultured at 37°C (containing 5% carbon dioxide). When the cell culture reached 80% fullness, the cells were transplanted. Remove the cell culture medium and add phosphate buffer to wash the cells. After removing the phosphate buffer, add 0.25% trypsin/2.21mMEDTA. After 20 minutes of reaction, add MEM-α containing 5% fetal calf serum to neutralize the pancreas. protease, collect the suspended cells, centrifuge, count the cells, and dilute them with MEM-α containing 5% fetal calf serum to a final concentration of ^5x10 cells per ml for subsequent subculture or For analytical purposes.

Example 2: Mitochondria Extraction

Culture human adipose stem cells until the cell number is ^1.5x108 cells. Wash the cells with Dulbecco's buffer solution (DPBS), remove the Dulbecco's buffer solution, add trypsin and react for 3 minutes, then add stem cell culture medium (KeratinocyteSFM (1X) liquid , bovine pituitary extract, 10wt% fetal bovine serum) to terminate the reaction, then collect the cells and centrifuge (600g, 10 minutes), remove the supernatant, and add 80 ml of IBC-1 buffer (buffer (225mM mannitol) , 75mM sucrose, 0.1mM EDTA, 30mM Tris-HCl pH 7.4) into the cells, homogenize and centrifuge, the resulting precipitate is mitochondria (hereinafter referred to as mitochondrial precipitate). Add 1.5 ml of IBC-1 to the mitochondrial precipitate buffer and proteolytic enzyme inhibitors and placed at 4°C for use in the following examples.

Example 3: Kidney epithelial cell damage test (1)

The kidney epithelial cells cultured in Example 1 were subcultured in a 96-well plate, where the concentration of each well was 5x10 ⁴ cells/200 μL. After 8 hours of culture, the supernatant was removed and washed with phosphate buffer. Add 200 μL of cell culture medium without 5% fetal bovine serum to each well and culture for 8 hours. After culture, different concentrations of hydrogen peroxide (0.3, 0.5, 1, 3, 5, 10mM) were given for treatment. After incubating with different concentrations of hydrogen peroxide for 24 hours, remove the supernatant from each well, then add cell culture medium containing 10% Alamar blue (100 μL/well), and incubate for 3-4 hours. , perform fluorescence signal measurement (Excitation/Emission: 560/590nm), and the results are shown in Figure 1A.

It can be seen from the results in Figure 1A that after the kidney epithelial cells are treated with different concentrations of hydrogen peroxide for 24 hours, the kidney epithelial cells will begin to be damaged. Among them, when the hydrogen peroxide concentration is above 1mM, the kidney epithelial cells will be damaged due to The situation leading to death increased significantly. Specifically, after treating kidney epithelial cells with hydrogen peroxide at a concentration of 1mM for 24 hours, the survival rate of kidney epithelial cells was 81.4%; treating kidney epithelial cells with hydrogen peroxide at a concentration of 5mM. After 24 hours, the survival rate of kidney epithelial cells was 31.3%; after treating kidney epithelial cells with hydrogen peroxide at a concentration of 10mM for 24 hours, the survival rate of kidney epithelial cells was 24.2%. The results show that treating renal epithelial cells with hydrogen peroxide can indeed establish a pattern of renal epithelial cell damage and death, and that as the concentration of added hydrogen peroxide increases, the damage to renal epithelial cells worsens and The number of kidney epithelial cell death also increased.

Example 4: Kidney epithelial cell damage test (2)

The kidney epithelial cells cultured in Example 1 were subcultured in a 96-well plate, where the concentration of each well was 1x10 ⁴ cells/200 μL. After 8 hours of culture, the supernatant was removed and washed with phosphate buffer. Add a volume of 200 μL of cell culture medium without 5% fetal calf serum to each well and incubate for 8 hours, then treat with hydrogen peroxide at concentrations of 1mM and 3mM for 4 hours, and then treat cells with different concentrations of hydrogen peroxide. Different doses (15 μg and 40 μg) of mitochondrial precipitate (prepared in Example 2) were cultured for 24 hours. After the culture, the supernatant was removed, and then cell culture medium containing 10% Alma Blue (100 μL/well) was added. , after culturing at 37°C for 3-4 hours, the fluorescence signal was measured (Excitation/Emission: 560/590nm) at the end of the culture, and the results are shown in Figure 1B.

It can be seen from the results in Figure 1B that kidney epithelial cells treated with hydrogen peroxide will be damaged, resulting in a decrease in cell survival rate. When the kidney epithelial cells have been damaged induced by hydrogen peroxide, a certain amount of mitochondrial precipitate is administered. , can significantly improve the survival rate of renal epithelial cells, and as the dose increases, the survival rate of renal epithelial cells also increases.

It can be seen from the results that the mitochondrial extract provided by the present invention can indeed protect kidney epithelial cells, avoid kidney epithelial cell damage caused by oxidation or inflammatory reactions, and can repair damaged kidney epithelial cells, effectively preventing kidney epithelial cells from being damaged. Cell death. In other words, the mitochondrial extract or composition containing the same provided by the present invention does have the effect of improving or/and preventing kidney damage or kidney disease caused by oxidative stress.

Example 5: Renal epithelial cell fibrosis test (1)

The kidney epithelial cells cultured in Example 1 were cultured in a 6-well plate with a culture medium containing 5% fetal bovine serum. The concentration of each well was 1x10 ⁵ cells/2ml. After 24 hours of culture, the supernatant was removed and then infused with phosphate. After washing with buffer, add 1 ml of cell culture medium without 5% fetal bovine serum to each well and culture for 8 hours, then add different concentrations of glycation end products (AGEs-BSA) (AGEs-BSA) at different concentrations (100 μg/ml and 400 μg/ml) and culture for 4 hours. , after the culture is completed, remove the cell culture medium containing AGEs-BSA and wash it with phosphate buffer saline, then add 1 ml of cell culture medium without 5% fetal bovine serum to each well and culture it for 24 and 48 hours respectively. The culture is completed. Afterwards, the supernatants were collected respectively, and collagen secretion was detected using a soluble collagen assay kit (Sircol ^TM Soluble Collagen Assay Kit). The results are shown in Figure 2A.

The results in Figure 2A show that whether kidney epithelial cells are treated with high concentration (400 μg/ml) or low concentration (100 μg/ml) of AGEs-BSA, the secretion and expression of collagen in kidney epithelial cells will be increased, and the amount of collagen will be increased. The secretion amount will increase with the treatment time, showing that AGEs-BSA can indeed induce renal epithelial cell lesions and produce fibrosis, which is the early stage of chronic kidney disease. If the secretion amount of collagen continues to increase, It will lead to the occurrence of chronic kidney disease.

Example 6: Renal epithelial cell fibrosis test (2)

The process of this example is generally the same as that of Example 5. The difference is that after removing the cell culture medium containing AGEs-BSA, cell culture medium without 5% fetal bovine serum and different doses (15 μg) are added to each well. and 40 μg) of mitochondrial precipitate (prepared in Example 2), and then cultured for 24 hours respectively. After the culture was completed, the supernatant was collected respectively, and a water-soluble collagen assay kit was used to detect collagen secretion. The results are shown in Figure 2B shown.

It can be seen from the results in Figure 2B that administration of mitochondrial precipitates can reduce the amount of collagen secretion induced by AGEs-BSA in renal epithelial cells, indicating that the mitochondria provided by the present invention or the composition containing them can effectively improve or/ and the efficacy of preventing renal fibrosis or related kidney diseases.

Example 7: Kidney epithelial cell fibrosis test (3)

The process of this example is generally the same as that of Example 6, except that the stimulant for inducing renal epithelial cell fibrosis is changed from AGEs-BSA to hydrogen peroxide; the detection results are shown in Figure 2C.

It can be seen from the results in Figure 2C that treating kidney epithelial cells with hydrogen peroxide will induce an increase in collagen secretion, which means that treating kidney epithelial cells with hydrogen peroxide can indeed construct a pattern of renal cell fibrosis; while giving hydrogen peroxide The processed mitochondrial sedimentation of renal epithelial cells can significantly reduce the amount of collagen secreted in the renal epithelial cells, indicating that the mitochondria provided by the present invention or the composition containing them can effectively improve or/and prevent renal fibrosis or cooperate with related to kidney disease.

Example 8: Test on mitochondrial function damage in renal epithelial cells

The kidney epithelial cells cultured in Example 1 were subcultured in a 96-well plate with cell culture medium containing 5% fetal bovine serum. The concentration of each well was 5x10 ⁴ cells/200 μL. After 24 hours of culture, the supernatant was removed. , after washing with phosphate buffer, add 1ml of cell culture medium without 5% fetal bovine serum to each well and culture for 8 hours. After culture, 3mM hydrogen peroxide and 100μg/ml AGEs-BSA were added to each well and cultured for 4 hours. , remove the cell culture medium containing hydrogen peroxide or AGEs-BSA, and wash it with phosphate buffer saline, then add 1 ml of cell culture medium without 5% fetal calf serum and different doses (15 μg and 40 μg) to each well. The mitochondrial precipitate (prepared in Example 2) was cultured for 24 hours respectively. After the culture was completed, it was washed with phosphate buffer, and then a buffer containing 10 μM JC-1 staining reagent was added, and the reaction was carried out at 37°C. After 10 minutes of cleaning, the fluorescence signal was measured (Excitation/Emission: 488/530nm). The results are shown in Figure 3.

It can be seen from the results in Figure 3 that only kidney epithelial cells treated with hydrogen peroxide or AGEs-BSA increased the expression of JC-1 monomer, indicating that the mitochondria in kidney epithelial cells were damaged by hydrogen peroxide or AGEs-BSA. The inflammatory environment caused by BSA leads to damage; in kidney epithelial cells treated first with hydrogen peroxide or AGEs-BSA, and then with different doses of mitochondrial precipitates, the expression of JC-1 monomers in them decreased significantly, and as the The expression of JC-1 monomers in renal epithelial cells decreased with increasing doses of mitochondrial precipitates.

Example 9: Animal testing

Take 10-week-old C57BL/6 mice and raise them in a constant temperature and humidity environment with a 12:12 hour light and dark cycle; the mouse kidney injury model will be ischemia-reperfusion (hereinafter referred to as I). /R kidney injury mode), the steps are as follows: first inject 150 mg/Kg of phenobarbital into the abdomen of the mouse by intraperitoneal injection, and then perform surgery on the left kidney of the mouse after the mouse becomes comatose. The left kidney was moved from the incision to the outside, and then the renal artery flowing into the kidney was blocked with a vascular clamp. After 30 minutes of occlusion, the vascular clamp was removed to allow the blood flow to pass again, and the I/R kidney injury mode was completed. After being treated with the R kidney injury model, different doses of mitochondria (15 μg and 40 μg) were delivered to the kidneys through renal artery vascular injection, namely the mitochondria high-dose group and the mitochondria low-dose group; the control group (I/R group) Inject phosphate buffered saline. After the mice in each group completed their treatment, the kidneys were moved back into the body and the wounds were sutured; blood samples were collected on the first day (D1) and the second day (D2) of the mice in each group, and serum creatinine was measured. (Creatinine, Cr) and blood urea nitrogen (BUN); then the extracted blood is centrifuged, the serum is separated and collected, and the contents of urea nitrogen and creatinine in the serum are analyzed; among them, serum creatinine is detected using mice The creatinine analysis kit (brand: Crystal Chem; model: 80350) was used for analysis; the serum urea nitrogen detection was analyzed with the urea analysis kit (brand: abcam; model: ab83362). After the mice in each group were sacrificed on the 7th day after transplantation (D7), the left kidney of each mouse was perfused and fixed in formalin, then paraffin embedded and tissue sectioned, and then H&E stained. The staining results were analyzed according to Histology studies the morphological changes caused by ischemic injury, and uses the Jablonski semi-quantitative standard to score the degree of renal damage: 0 points, normal tissue; 1 point, renal tubular damage area is less than 5%; 2 points, renal tubule damage area is more than 5% to less than 25%; 3 points, the area of renal tubular damage is greater than 25% to less than 75%; 4 points, the area of renal tubular damage is greater than 75%.

The above results are shown in Table 1 below. From the results in Table 1, it can be seen that compared with the control group, the expression levels of urea nitrogen and creatinine in the serum of the I/R group will increase significantly, showing that the I/R kidney injury model does cause kidney damage, and , from the results of the kidney damage score, it can be known that the score of the I/R group is 3-4 points, which represents serious renal tubular damage; and compared with the I/R group, the mice in the group given mitochondria had less urea in the serum The contents of nitrogen and creatinine were significantly reduced, showing that giving mitochondria can effectively improve kidney damage. The results of the kidney damage score show that giving mitochondria can restore damaged kidney cells and improve the condition of renal tubular damage. With the administration of mitochondria, As the dose increases, the effect of improving kidney damage increases.

Table 1: Analysis results of serum and kidney tissue sections of mice in each group after different treatments

The above results show that when kidney cells are damaged due to fibrosis, oxidative stress or inflammatory environment, administering the mitochondrial extract provided by the present invention or a composition containing the same can effectively improve the mitochondrial damage of kidney cells. Therefore, it can achieve the effect of improving or treating kidney cell damage or related diseases.

Claims (6)

1. Use of mitochondria for the preparation of a composition for reducing the amount of collagen secretion from the kidneys.

2. The use of claim 1, wherein an increase in the amount of collagen secreted by the kidney causes kidney fibrosis.

3. The use according to claim 2, wherein the kidney fibrosis is caused by damage to kidney epithelial cells.

4. The use according to claim 1, wherein the mitochondrial dose in the composition is above 5 μg.

5. The use according to claim 1, wherein the mitochondrial dose in the composition is above 40 μg.

6. The use according to claim 1, wherein the concentration of mitochondria in the composition is 75-200 μg/ml.